The emergence of AIDS as a disease spread through sexual intercourse has prompted the search for new, effective, safe, and female-controlled vaginal microbicides for curbing mucosal viral transmission. Microbicides would provide protection by inactivating viruses or preventing viruses from replicating either in semen or the infected host cells that line the vaginal wall. Through an integrated effort involving synthesis, docking studies, and biological evaluation, we identified a thiophene thiourea non-nucleoside inhibitor (N'-[2-(2-thiophene)ethyl]-N'-[2-(5-bromopyridyl)] thiourea [PHI-443]) with unprecedented potency against genotypic and phenotypic drug-resistant HIV-1 strains. PHI-443 had no effect on human sperm functions even at a concentration 30,000-times higher than it in vitro anti-HIV activity. PHI-443 showed favorable pharmacokinetics and did not cause acute or subacute toxicity in mice. The discovery of PHI-443 as a non-spermicidal, non-toxic, broad-spectrum anti-HIV agent represents a significant step forward in the development of a microbicide for curbing heterosexual HIV transmission. Non-spermicidal anti-HIV agents that are highly active against genotypic and phenotypic drug-resistant primary clinical HIV-1 isolates will be a tremendous advantage for the development of a broad-spectrum anti-HIV microbicide since a high percentage of newly infected individuals in the US harbor drug-resistant mutants. As a non-spermicidal prophylactic microbicide, PHI-443 will be useful for (i) sexually active women to allow pregnancy while protecting both mother and child from HIV and; (ii) as a prophylactic antiviral agent, especially for HIV-1 serodiscordant couples before assisted reproductive technology procedures are undertaken. The goals of this Phase I proposal are: (i) to examine the microbicidal activity of PHI-443 containing gel-microemulsion in vaginal HIV-1 infected Hu-PBL-SCID mouse model of sexually transmitted AIDS; and (ii) to perform pilot studies aimed at the evaluation of the tissue compatibility, safety, and pharmacokinetic analysis of PHI-443 gel formulation in the rabbit model. Specific Aims 1 and 2 may provide the foundation for the clinical development of PHI-443 in Phase II studies as a safe, effective broad-spectrum anti-HIV microbicide without conception-inhibiting functions.